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The importance of Chromatin Immunoprecipitation (ChIP) technology has grown exponentially along with an increased interest in epigenetic regulation. The correlation of transcription factors with histone marks is now well established as the center of epigenetic studies; therefore, precise knowledge about histone marks is critical to unravel their molecular function and to understand their role in biological systems. This knowledge constantly accumulates and is provided openly in the expanding hubs of information such as the USCS Genome Browser. Nevertheless, as we gain more knowledge, we realize that the DNA-protein interactions are not driven by a "one size fits all" rule. Also, the diversity of interactions between DNA, histones, and transcriptional regulators is much bigger than previously considered. Besides a detailed protocol of sample preparation for the ChIP assay from primary human monocyte-derived macrophages (MDM) [an acceptable in vitro model for primary, human macrophage cells], we show that differences between various types of cells exist. Furthermore, we can postulate that such variations exist between transformed macrophage-like cell lines and primary macrophages obtained from healthy volunteers. We found that the most efficient fixation time for MDM is 10min. Finally, to perform multiple analytical assays, we showed that even with thorough methodology, the yield of material obtained from primary cells is the major challenge.
The transcriptional regulatory network in prokaryotes controls global gene expression mostly through transcription factors (TFs), which are DNA-binding proteins. Chromatin immunoprecipitation (ChIP) with DNA sequencing methods can identify TF binding sites across the genome, providing a bottom-up, mechanistic understanding of how gene expression is regulated. ChIP provides indispensable evidence toward the goal of acquiring a comprehensive understanding of cellular adaptation and regulation, including condition-specificity. ChIP-derived data's importance and labor-intensiveness motivate its broad dissemination and reuse, which is currently an unmet need in the prokaryotic domain. To fill this gap, we present proChIPdb (prochipdb.org), an information-rich, interactive web database. This website collects public ChIP-seq/-exo data across several prokaryotes and presents them in dashboards that include curated binding sites, nucleotide-resolution genome viewers, and summary plots such as motif enrichment sequence logos. Users can search for TFs of interest or their target genes, download all data, dashboards, and visuals, and follow external links to understand regulons through biological databases and the literature. This initial release of proChIPdb covers diverse organisms, including most major TFs of Escherichia coli, and can be expanded to support regulon discovery across the prokaryotic domain.
Chromatin-immunoprecipitation (ChIP) employs generally a mild formaldehyde cross-linking step, which is followed by isolation of specific protein-DNA complexes and subsequent PCR testing, to analyze DNA-protein interactions. Poly(ADP-ribosyl)ation, a posttranslational modification involved in diverse cellular functions like repair, replication, transcription, and cell death regulation, is most prominent after DNA damage. Poly(ADP-ribose)polymerase-1 is activated upon binding to DNA strand-breaks and coordinates repair by recruitment or displacement of proteins. Several proteins involved in different nuclear pathways are directly modified or contain poly(ADP-ribose)-interaction motifs. Thus, poly(ADP-ribose) regulates chromatin composition. In immunofluorescence experiments, we noticed artificial polymer-formation after formaldehyde-fixation of undamaged cells. Therefore, we analyzed if the formaldehyde applied during ChIP also induces poly(ADP-ribosyl)ation and its impact on chromatin composition. We observed massive polymer-formation in three different ChIP-protocols tested independent on the cell line. This was due to induction of DNA damage signaling as monitored by γH2AX formation. To abrogate poly(ADP-ribose) synthesis, we inhibited this enzymatic reaction either pharmacologically or by increased formaldehyde concentration. Both approaches changed ChIP-efficiency. Additionally, we detected specific differences in promoter-occupancy of tested transcription factors as well as the in the presence of histone H1 at the respective sites. In summary, we show here that standard ChIP is flawed by artificial formation of poly(ADP-ribose) and suppression of this enzymatic activity improves ChIP-efficiency in general. Also, we detected specific changes in promoter-occupancy dependent on poly(ADP-ribose). By preventing polymer synthesis with the proposed modifications in standard ChIP protocols it is now possible to analyze the natural chromatin-composition.
We report on a detailed chromatin immunoprecipitation (ChIP) protocol for the unicellular green alga Chlamydomonas reinhardtii. The protocol is suitable for the analysis of nucleosome occupancy, histone modifications and transcription factor binding sites at the level of mononucleosomes for targeted and genome-wide studies. We describe the optimization of conditions for crosslinking, chromatin fragmentation and antibody titer determination and provide recommendations and an example for the normalization of ChIP results as determined by real-time PCR.
With the advent of next generation high-throughput DNA sequencing technologies, omics experiments have become the mainstay for studying diverse biological effects on a genome wide scale. Chromatin immunoprecipitation (ChIP-seq) is the omics technique that enables genome wide localization of transcription factor (TF) binding or epigenetic modification events. Since the inception of ChIP-seq in 2007, many methods have been developed to infer ChIP-target binding loci from the resultant reads after mapping them to a reference genome. However, interpreting these data has proven challenging, and as such these algorithms have several shortcomings, including susceptibility to false positives due to artifactual peaks, poor localization of binding sites and the requirement for a total DNA input control which increases the cost of performing these experiments. We present Ritornello, a new approach for finding TF-binding sites in ChIP-seq, with roots in digital signal processing that addresses all of these problems. We show that Ritornello generally performs equally or better than the peak callers tested and recommended by the ENCODE consortium, but in contrast, Ritornello does not require a matched total DNA input control to avoid false positives, effectively decreasing the sequencing cost to perform ChIP-seq. Ritornello is freely available at https://github.com/KlugerLab/Ritornello.
HMGA2 is an architectonic transcription factor abundantly expressed during embryonic and fetal development and it is associated with the progression of malignant tumors. The protein harbours three basically charged DNA binding domains and an acidic protein binding C-terminal domain. DNA binding induces changes of DNA conformation and hence results in global overall change of gene expression patterns. Recently, using a PCR-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure two consensus sequences for HMGA2 binding have been identified.
Extensive sonication of formaldehyde-crosslinked chromatin can generate DNA fragments averaging 200 bp in length (range 75-300 bp). Fragmentation is largely random with respect to genomic region and nucleosome position. ChIP experiments employing such extensively fragmented samples show 2- to 4-fold increased enrichment of protein binding sites over control genomic regions, when compared to samples sonicated to a more conventional size range (300-500 bp). The basis of improved fold enrichments is that immunoprecipitation of protein-bound regions is unaffected by fragment size, whereas immunoprecipitation of control genomic regions decreases progressively along with reduced fragment size due to fewer nonspecific binding sites. The use of extensively sonicated samples improves mapping of protein binding sites, and it extends the dynamic range for quantitative measurements of histone density. We show that many yeast promoter regions are virtually devoid of histones.
Chromatin immunoprecipitation (ChIP) analysis is widely used to identify the locations in genomes occupied by transcription factors (TFs). The approach involves chemical cross-linking of DNA with associated proteins, fragmentation of chromatin by sonication or enzymatic digestion, immunoprecipitation of the fragments containing the protein of interest, and then PCR or hybridization analysis to characterize and quantify the genomic sequences enriched. We developed a computational model of quantitative ChIP analysis to elucidate the factors contributing to the method's resolution. The most important variables identified by the model were, in order of importance, the spacing of the PCR primers, the mean length of the chromatin fragments, and, unexpectedly, the type of fragment width distribution, with very small DNA fragments and smaller amplicons providing the best resolution of TF binding. One of the major predictions of the model was also validated experimentally.
The growth of sequencing-based Chromatin Immuno-Precipitation studies call for a more in-depth understanding of the nature of the technology and of the resultant data to reduce false positives and false negatives. Control libraries are typically constructed to complement such studies in order to mitigate the effect of systematic biases that might be present in the data. In this study, we explored multiple control libraries to obtain better understanding of what they truly represent.
The small ubiquitin-like modifier (SUMO) is a short peptide that can be covalently linked to proteins altering their function. SUMOylation is an essential post-translational modification (PTM). Because of its dynamic nature, low abundance levels, and technical limitations, the occupation of endogenous SUMOylated transcription factors at genomic loci is challenging to detect. The chromatin regulator Methyl CpG binding protein 2 (MeCP2) is subjected to PTMs including SUMO. Mutations in MeCP2 lead to Rett syndrome, a severe neurodevelopmental disorder. Here, we present an efficient method to perform sequential chromatin immunoprecipitation (Seq-ChIP) for detecting SUMOylated MeCP2 in neurons. This Seq-ChIP technique is a useful tool to determine the occupancy of SUMOylated transcription and chromatin factors at specific genomic regions.
Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. However, non-biased methods to identify molecules bound to specific genomic loci in vivo are limited. Here, we applied insertional chromatin immunoprecipitation (iChIP) to direct identification of components of insulator complexes, which function as boundaries of chromatin domain. We found that the chicken β-globin HS4 (cHS4) insulator complex contains an RNA helicase protein, p68/DDX5; an RNA species, steroid receptor RNA activator 1; and a nuclear matrix protein, Matrin-3, in vivo. Binding of p68 and Matrin-3 to the cHS4 insulator core sequence was mediated by CCCTC-binding factor (CTCF). Thus, our results showed that it is feasible to directly identify proteins and RNA bound to a specific genomic region in vivo by using iChIP.
Several recent studies have shown a genetic influence on gene expression variation, including variation between the two chromosomes within an individual and variation between individuals at the population level. We hypothesized that genetic inheritance may also affect variation in chromatin states. To test this hypothesis, we analyzed chromatin states in 12 lymphoblastoid cells derived from two Centre d'Etude du Polymorphisme Humain families using an allele-specific chromatin immunoprecipitation (ChIP-on-chip) assay with Affymetrix 10K SNP chip. We performed the allele-specific ChIP-on-chip assays for the 12 lymphoblastoid cells using antibodies targeting at RNA polymerase II and five post-translation modified forms of the histone H3 protein. The use of multiple cell lines from the Centre d'Etude du Polymorphisme Humain families allowed us to evaluate variation of chromatin states across pedigrees. These studies demonstrated that chromatin state clustered by family. Our results support the idea that genetic inheritance can determine the epigenetic state of the chromatin as shown previously in model organisms. To our knowledge, this is the first demonstration in humans that genetics may be an important factor that influences global chromatin state mediated by histone modification, the hallmark of the epigenetic phenomena.
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a method designed to detect interactions between chromatin and the proteins bound to it. This method has been widely used for characterizing epigenetic landscapes in many cell types; however, a limiting factor has been the requirement of a high number of cells. Here, we describe a protocol for ChIP in epidermal cells from a newborn mouse, purified by fluorescence-activated cell sorting (FACS). This protocol has been optimized specifically for prefixed, low cell numbers, resulting in enough immunoprecipitated DNA suitable for genome-wide analysis.
Chromatin immunoprecipitation and deep sequencing (ChIP-SEQ) represents a powerful tool for identifying the genomic targets of transcription factors, chromatin remodeling factors, and histone modifications. The frogs Xenopus laevis and Xenopus tropicalis have historically been outstanding model systems for embryology and cell biology, with emerging utility as highly accessible embryos for genome-wide studies. Here we focus on the particular strengths and limitations of Xenopus cell biology and genomics as they apply to ChIP-SEQ, and outline a methodology for ChIP-SEQ in both species, providing detailed strategies for sample preparation, antibody selection, quality control, sequencing library preparation, and basic analysis.
Transcriptional regulation studies of CNS neurons are complicated by both cellular diversity and plasticity. Microdissection of specific functionally related populations of neurons can greatly reduce these issues, but typically excludes the use of many technologies due to tissue requirements, such as Chromatin Immunoprecipitation (ChIP), a powerful tool for studying in vivo protein-DNA interactions. We have developed a fast carrier ChIP (Fast CChIP) method for analyzing specific in vivo transcription factor-DNA interactions in as little as 0.2 mm(3) brain tissue. Using an antibody against phosphorylated cyclic-AMP response element binding (CREB) protein, we confirmed phospho-CREB (pCREB) binding at the c-fos gene promoter. Then we further demonstrated the applicability of Fast CChIP in determining hypertension-induced pCREB binding at the c-fos gene promoter in the rat nucleus tractus solitarius (NTS), confirming CREB's role in mediating hypertension-induced c-fos expression. This method will be broadly applicable to individual brain nucleus and biopsy/surgical samples.
Existing monitoring approaches in heart transplantation lack the sensitivity to provide deep molecular assessments to guide management, or require endomyocardial biopsy, an invasive and blind procedure that lacks the precision to reliably obtain biopsy samples from diseased sites. This study examined plasma cell-free DNA chromatin immunoprecipitation sequencing (cfChIP-seq) as a noninvasive proxy to define molecular gene sets and sources of tissue injury in heart transplant patients. In healthy controls and in heart transplant patients, cfChIP-seq reliably detected housekeeping genes. cfChIP-seq identified differential gene signals of relevant immune and nonimmune molecular pathways that were predominantly down-regulated in immunosuppressed heart transplant patients compared with healthy controls. cfChIP-seq also identified cell-free DNA tissue sources. Compared with healthy controls, heart transplant patients demonstrated greater cell-free DNA from tissue types associated with heart transplant complications, including the heart, hematopoietic cells, lungs, liver, and vascular endothelium. cfChIP-seq may therefore be a reliable approach to profile dynamic assessments of molecular pathways and sources of tissue injury in heart transplant patients.
The processes that compose expression of a given gene are far more complex than previously thought presenting unprecedented conceptual and mechanistic challenges that require development of new tools. Chromatin structure, which is regulated by DNA methylation and histone modification, is at the center of gene regulation. Immunoprecipitations of chromatin (ChIP) and methylated DNA (MeDIP) represent a major achievement in this area that allow researchers to probe chromatin modifications as well as specific protein-DNA interactions in vivo and to estimate the density of proteins at specific sites genome-wide. Although a critical component of chromatin structure, DNA methylation has often been studied independently of other chromatin events and transcription.
Chromatin immunoprecipitation (ChIP) assay is widely used for investigating the interaction between DNA and DNA-binding proteins such as transcription factors, co-factors, or chromatin-associated proteins. However, a successful ChIP assay largely depends on the quality of a ChIP-grade primary antibody. In cases where specific antibodies are unavailable or with low binding affinity, here, we describe a tailored protocol to achieve robust and reproducible chromatin binding by expressing an exogenous epitope-tagged protein in cells, followed by ChIP assays using a tag-specific antibody. For complete details on the use and execution of this protocol, please refer to Fang et al. (2021)1 and Kidder et al. (2011).2.
High-throughput, microarray-based chromatin immunoprecipitation (ChIP-chip) technology allows in vivo elucidation of transcriptional networks. However this complex is not yet readily accessible, in part because its many parameters have not been systematically evaluated and optimized. We address this gap by systematically assessing experimental-design parameters including antibody purity, dye-bias, array-batch, inter-day hybridization bias, amplification method and choice of hybridization control. The combined performance of these optimized parameters shows a 90% validation rate in ChIP-chip analysis of Myc genomic binding in HL60 cells using two different microarray platforms. Increased sensitivity and decreased noise in ChIP-chip assays will enable wider use of this methodology to accurately and affordably elucidate transcriptional networks.
Transcription factors are important regulators of cell fate and function. Knowledge about where transcription factors are bound in the genome is crucial for understanding their function. A common method to study protein-DNA interactions is chromatin immunoprecipitation (ChIP). Here, we present a revised ChIP protocol to determine protein-DNA interactions for the yeast Saccharomyces cerevisiae. We optimized several aspects of the procedure, including cross-linking and quenching, cell lysis, and immunoprecipitation steps. This protocol facilitates sensitive and reproducible quantitation of protein-DNA interactions. For complete details on the use and execution of this protocol, please refer to (de Jonge et al., 2019).
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